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Variant Calling · FreedomIntelligence | DecimalAI

Bio Variant Calling

SkillScore9Maintained

bio-variant-calling

Call SNPs and indels from aligned reads using bcftools mpileup and call. Use when detecting variants from BAM files or generating VCF from alignments.

scienceby FreedomIntelligence

Links this skill to your agents and keeps it current with the author’s updates.

Automatic

Research pack

1 · One-time — install the CLI (docs ↗)

pip install decimalai

2 · Get the skill — writes .claude/skills/bio-variant-calling/SKILL.md

decimalai skills pull bio-variant-calling --out .claude/skills/

No CLI? Get the same file — curl · raw file

curl -fsSL --create-dirs -o .claude/skills/bio-variant-calling/SKILL.md https://app.decimal.ai/s/bio-variant-calling/SKILL.md

Raw file: /s/bio-variant-calling/SKILL.md ↗ · machine-readable summary at /s/bio-variant-calling.json ↗

In productionGlobal

how this skill behaves on real traffic · last 30 days

Runs

all versions · 30d

Anonymized aggregate across every org using this skill — proof from real production use, not a demo.

Does this skill help? · Benchmark: with vs. withoutsign in to run this on your agents

22 test cases from the publisher’s eval.yaml · each run twice on gemini-3.6-flash — with the skill injected, then without · last run 57d ago

Yes — +9% lift · at higher cost+144% tokens0% turns9% of whole cases pass with skill (2/22)

Test case	Without → With	Effect	Δ tokens	Δ turns
case-20	✗→✓	▲ Improved	—	—
case-13	✗→✓	▲ Improved	—	—
case-02	✗→✗	＝ Same ✗	—	—
case-03	✗→✗	＝ Same ✗	—	—
case-21	✗→✗	＝ Same ✗	—	—

Showing 5 of 22 cases

SkillSafety

✓ 3 of 3 passed

Code scan15 of 15 checks clean

0 critical · 0 warning · 0 info — scanner v4 — 7/30/2026

live_secretremote_code_executionreverse_shelldata_exfiltrationssrf_metadataagent_snoopinganti_refusaldestructive_commandsobfuscated_unicodeunicode_homoglyphopaque_blobsuspicious_urltool_overreachtrigger_abuseprompt_injection_phrasing

IntentSafe

LLM judge · does the skill do what it claims

ContentClean

LLM judge · policy & harmful-content review

Source

Imported

FreedomIntelligence / OpenClaw-Medical-Skills

skills/bio-variant-calling/SKILL.md

View on GitHub ↗

Imported 8/1/2026Version v1

Skill contents

SKILL.md

Version Compatibility

Reference examples tested with: bcftools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Variant Calling

Call SNPs and indels from aligned reads using bcftools.

Basic Workflow

BAM file + Reference FASTA
         |
         v
   bcftools mpileup (generate pileup)
         |
         v
   bcftools call (call variants)
         |
         v
   VCF file

bcftools mpileup + call

Goal: Detect SNPs and indels from aligned reads using the bcftools pileup-and-call pipeline.

Approach: Generate per-position pileup likelihoods with mpileup, then call genotypes with the multiallelic caller.

"Call variants from my BAM file" → Generate genotype likelihoods from aligned reads and identify variant sites using a Bayesian caller.

Basic Variant Calling

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -o variants.vcf

Output Compressed VCF

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -Oz -o variants.vcf.gz
bcftools index variants.vcf.gz

Call Specific Region

bash
bcftools mpileup -f reference.fa -r chr1:1000000-2000000 input.bam | \
    bcftools call -mv -o region.vcf

Call from Multiple BAMs

bash
bcftools mpileup -f reference.fa sample1.bam sample2.bam sample3.bam | \
    bcftools call -mv -o variants.vcf

BAM List File

bash
# bams.txt: one BAM path per line
bcftools mpileup -f reference.fa -b bams.txt | bcftools call -mv -o variants.vcf

mpileup Options

Goal: Control pileup generation with quality thresholds, annotations, and region restrictions.

Approach: Set minimum mapping/base quality, request specific FORMAT/INFO tags, and restrict to target regions.

Quality Filtering

bash
bcftools mpileup -f reference.fa \
    -q 20 \           # Min mapping quality
    -Q 20 \           # Min base quality
    input.bam | bcftools call -mv -o variants.vcf

Annotate with Read Depth

bash
bcftools mpileup -f reference.fa -a DP,AD input.bam | bcftools call -mv -o variants.vcf

Full Annotation Set

bash
bcftools mpileup -f reference.fa \
    -a FORMAT/DP,FORMAT/AD,FORMAT/ADF,FORMAT/ADR,INFO/AD \
    input.bam | bcftools call -mv -o variants.vcf

Target Regions (BED)

bash
bcftools mpileup -f reference.fa -R targets.bed input.bam | \
    bcftools call -mv -o variants.vcf

Max Depth

bash
bcftools mpileup -f reference.fa -d 1000 input.bam | bcftools call -mv -o variants.vcf

call Options

Calling Models

| Flag | Model | Use Case | |------|-------|----------| | -m | Multiallelic caller | Default, recommended | | -c | Consensus caller | Legacy, single sample |

Output Variants Only

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -o variants.vcf
# -v outputs variant sites only (not reference calls)

Output All Sites

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -m -o all_sites.vcf
# Without -v, outputs all sites including reference

Ploidy

bash
# Haploid calling
bcftools mpileup -f reference.fa input.bam | bcftools call -m --ploidy 1 -o variants.vcf

# Specify ploidy file
bcftools mpileup -f reference.fa input.bam | bcftools call -m --ploidy-file ploidy.txt -o variants.vcf

Prior Probability

bash
# Adjust variant prior (default 1.1e-3)
bcftools mpileup -f reference.fa input.bam | bcftools call -m -P 0.001 -o variants.vcf

Common Pipelines

Goal: Run production-ready variant calling workflows for single-sample and multi-sample analyses.

Approach: Chain mpileup and call with quality filters, annotations, and compressed output, optionally parallelized by chromosome.

Standard SNP/Indel Calling

bash
bcftools mpileup -Ou -f reference.fa \
    -q 20 -Q 20 \
    -a FORMAT/DP,FORMAT/AD \
    input.bam | \
bcftools call -mv -Oz -o variants.vcf.gz

bcftools index variants.vcf.gz

Multi-sample Calling

bash
bcftools mpileup -Ou -f reference.fa \
    -a FORMAT/DP,FORMAT/AD \
    sample1.bam sample2.bam sample3.bam | \
bcftools call -mv -Oz -o cohort.vcf.gz

bcftools index cohort.vcf.gz

Calling with Regions

bash
bcftools mpileup -Ou -f reference.fa \
    -R targets.bed \
    -a FORMAT/DP,FORMAT/AD \
    input.bam | \
bcftools call -mv -Oz -o targets.vcf.gz

Parallel by Chromosome

bash
for chr in chr1 chr2 chr3; do
    bcftools mpileup -Ou -f reference.fa -r "$chr" input.bam | \
        bcftools call -mv -Oz -o "${chr}.vcf.gz" &
done
wait

# Concatenate results
bcftools concat -Oz -o all.vcf.gz chr*.vcf.gz
bcftools index all.vcf.gz

Annotation Tags

INFO Tags

| Tag | Description | |-----|-------------| | DP | Total read depth | | AD | Allelic depths | | MQ | Mapping quality | | FS | Fisher strand bias | | SGB | Segregation based metric |

FORMAT Tags

| Tag | Description | |-----|-------------| | GT | Genotype | | DP | Read depth per sample | | AD | Allelic depths per sample | | ADF | Forward strand allelic depths | | ADR | Reverse strand allelic depths | | GQ | Genotype quality | | PL | Phred-scaled likelihoods |

Request Specific Annotations

bash
bcftools mpileup -f reference.fa \
    -a FORMAT/DP,FORMAT/AD,FORMAT/SP,INFO/AD \
    input.bam | bcftools call -mv -o variants.vcf

Performance Options

Goal: Speed up variant calling for large datasets.

Approach: Use multi-threading and uncompressed BCF piping to reduce I/O overhead.

Multi-threading

bash
bcftools mpileup -f reference.fa --threads 4 input.bam | \
    bcftools call -mv --threads 4 -o variants.vcf

Uncompressed BCF for Speed

bash
bcftools mpileup -Ou -f reference.fa input.bam | bcftools call -mv -Ou | \
    bcftools filter -Oz -o filtered.vcf.gz

Quick Reference

| Task | Command | |------|---------| | Basic calling | bcftools mpileup -f ref.fa in.bam \| bcftools call -mv -o out.vcf | | With quality filter | bcftools mpileup -f ref.fa -q 20 -Q 20 in.bam \| bcftools call -mv | | Region | bcftools mpileup -f ref.fa -r chr1:1-1000 in.bam \| bcftools call -mv | | Multi-sample | bcftools mpileup -f ref.fa s1.bam s2.bam \| bcftools call -mv | | With annotations | bcftools mpileup -f ref.fa -a DP,AD in.bam \| bcftools call -mv |

Common Errors

| Error | Cause | Solution | |-------|-------|----------| | no FASTA reference | Missing -f | Add -f reference.fa | | reference mismatch | Wrong reference | Use same reference as alignment | | no variants called | Low quality/depth | Lower quality thresholds |

Related Skills

vcf-basics - View and query resulting VCF
filtering-best-practices - Filter variants by quality
variant-normalization - Normalize indels
alignment-files/pileup-generation - Alternative pileup generation

Illustrative Want to see what bio-variant-calling would put in front of your model? Preview the rendered body and check its trigger phrases — no LLM call, no signup.

Latest benchmark · publisher runVerified

7/26/2026, 09:55 UTC · 22 cases

Lift

+9%

9% vs 0%

Agent turns

1.0 vs 1.0

Tokens

+144%

2,960.18 vs 1,212.23

fail → pass · 2fail → fail · 20

Per-case results (22)

Case	Status	Duration (ms)			Turns			Tokens			Tool calls
Case	Status	Without	With	Δ	Without	With	Δ	Without	With	Δ	Without	With	Δ
case-02 I am calling variants from aligned_reads.bam against hg38.fa using bcftools. I want the final output file variants.vcf.gz to be directly bgzf-compressed during the call step without having to pipe through bgzip afterwards. Which output format flag should be passed to bcftools call?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-03 I want to call variants from alignment.bam and ref.fa strictly within chromosome region chr1:1000000-2000000 using bcftools. I am tempted to specify the region flag on the bcftools call step. Which command step should receive the region restriction flag?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-20 I already have an annotated raw call file named raw_variants.vcf.gz. I want to filter out low-quality calls by removing any variant site where QUAL is less than 30 or DP is less than 10, writing the passing sites to filtered.vcf.gz. What command line invocation should be used?	fail→pass	—	—	—	—	—	—	—	—	—	—	—	—
case-21 I have an existing variant call file variants.vcf containing complex indels and multiallelic records. I need to left-align indels and split multiallelic records against human reference hg38.fa using bcftools. What subcommand performs this VCF normalization?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-12 I am calling variants from a bacterial genome alignment E_coli.bam against ref.fasta using bcftools. Since the organism is haploid, default diploid calling assumptions will produce incorrect genotype calls. How do I specify haploid ploidy during calling?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-15 I am setting up a high-throughput variant calling pipeline where bcftools mpileup pipes directly into bcftools call. Piping plain text VCF causes severe bottlenecking. I want mpileup to emit uncompressed BCF output directly into the stream for speed. Which output type flag achieves this?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-10 I am running variant identification using bcftools call on pileup data. I want to use the modern multiallelic and rare-variant caller algorithm rather than the legacy consensus calling model. Which option flag selects this model?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-08 I am performing targeted exone variant calling on sample.bam using bcftools. The genomic target capture regions are defined in capture_targets.bed. I want to restrict candidate generation to these intervals. Which flag should be passed to bcftools mpileup?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-17 I ran variant calling in parallel across individual chromosomes, producing chr1.vcf.gz, chr2.vcf.gz, and chr3.vcf.gz. Now I want to merge these coordinate-sorted, non-overlapping genomic VCF files into a single genome-wide VCF file named all.vcf.gz using bcftools. Which subcommand is designed for concatenating sorted regional VCFs?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-18 To perform strand bias filtering downstream, I need bcftools mpileup to calculate forward and reverse strand allele depths for each sample. Which specific FORMAT tags must be requested in the annotation flag?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-07 I need to call variants from sample.bam using bcftools and ensure the resulting VCF includes per-sample total depth and allelic depth tags in the FORMAT field. What flag and tag specifications must be supplied to bcftools mpileup?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-01 I have an aligned alignment file sample.bam and reference genome ref.fa. I want to generate raw genotype likelihoods and call variant sites into out.vcf using standard C-based command line utilities. I am tempted to use samtools mpileup directly into bcftools view, but I know that pipeline is deprecated. What is the standard current command line pipeline?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-14 I am calling variants in a highly polymorphic viral population sample. I want to increase the prior probability of discovering a non-reference site in bcftools call to 0.001 instead of the default 0.0011. Which flag adjusts the mutation prior probability?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-13 I am analyzing sex chromosome variants in human samples where males and females have different ploidy settings across X and Y chromosomes. I have created sex_ploidy.txt. How do I instruct bcftools call to apply this ploidy mapping?	fail→pass	—	—	—	—	—	—	—	—	—	—	—	—
case-05 I have 50 BAM files listed one per line in a text file named cohort_bams.txt. I want bcftools to perform variant calling across all files in cohort_bams.txt without expanding them all on the command line prompt. What option flag reads BAM paths from a file?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-11 When executing bcftools call on genotype likelihoods, the output VCF includes every reference position evaluated. I want to restrict the output file strictly to candidate variant sites. Which flag enables variant-only output?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-06 When running variant detection on tumor.bam with ref.fa using bcftools mpileup, I want to ignore alignment reads with mapping quality below 20 and bases with quality score below 20. I am tempted to use lowercase -q for both thresholds. What are the distinct flags for mapping quality and base quality?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-22 I have a multi-sample calls file cohort.vcf.gz. I want to extract chromosome, position, reference allele, alternate allele, and sample genotypes into a tab-delimited text summary table without any VCF header lines. Which bcftools command produces custom formatted text output?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-04 I have three separate alignment files: sample1.bam, sample2.bam, and sample3.bam, along with genome.fa. I want to perform joint variant calling across all three samples simultaneously into joint.vcf using bcftools. How should the input BAM files be specified?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-16 I am processing a 50x whole genome BAM file with bcftools mpileup piped to bcftools call. The run is taking too long on single CPU core. I want to assign 4 parallel CPU worker threads to both mpileup and call commands. Which command line option specifies thread count?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-19 I am conducting a reference-guided population genomics study where invariant (homozygous reference) positions are required alongside variants to estimate genome-wide nucleotide diversity. How should I invoke bcftools call to retain non-variant reference positions?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—
case-09 In high-coverage amplicon sequencing regions, bcftools mpileup stalls due to default read depth limits. I want to set the maximum per-file depth cap to 1000 reads to prevent excessive memory usage. Which flag controls maximum depth in mpileup?	fail→fail	—	—	—	—	—	—	—	—	—	—	—	—

These cases were also screened by the skill’s intent / safety pass.See the Trust review ↗Reproduce this benchmark in Colab ↗

How this skill was measured

DecimalAI ran this skill against gemini-3.6-flash twice over the same eval suite — once with the skill loaded and once without — and compared the two runs case by case. 22 cases were attempted. The headline lift of +9 percentage points is the difference between those two pass rates over the 22 comparable cases.

Model measured: gemini-3.6-flash
Graded by: gemini-3.6-flash
Grading method: judged
Verification: verified
Pass rate without the skill: 0%
Pass rate with the skill: 9.1%
Cases attempted: 22
Cases counted toward lift: 22
Cases that regressed: 0
Skill version measured: v1
Run completed: 7/26/2026, 09:55 UTC

The per-case answers from this run were removed by the retention sweep, so the case table below shows the verdicts without the text either arm produced. The counts above were recorded at the time and are unaffected. Answers are now kept for 180 days.

SkillScore blends 4 signals1 of 4 complete

BenchmarkPass rate WITH skill on a verified run9

Live evalNeeds ≥10 evaluated traces in 30 days

AI ratingNeeds ≥10 rated traces

AdoptionNeeds ≥10 serves across ≥2 orgs in 30 days

Backed by 1 of 4 signals — each added signal makes the score more robust.

Verified on gemini-3.6-flash · re-tested 57 days ago

Other measured skills in the registry, with their headline benchmark lift.